The present invention relates to the enzymic assay for acetate ion in a liquid sample, particularly liquids of fruits juice or biological origin.
It is important to analyze for acetate ion in solutions of fruit juice origin such as wine. The acetic acid level in wine is important to the enologist and the wine maker as an indicator of quality control or spoilage. In one detection technique, the acetate ion is reacted with adenosine triphosphate (ATP) in the presence of acetate kinase (AK) to form acetyl-phosphate and adenosine diphosphate (ADP). Then the acetyl-phosphate is used as the basis for analysis in a colorimetric assay by wet chemistry. This assay technique suffers from the disadvantages of being slow, relatively inaccurate and only being adaptable to the assay of white wine.
Acetate esters such as ethyl acetate provide an objectionable taste to the consumer of wines. Therefore, a simple, efficient assay for such esters would be advantageous.
In clinical biochemistry, the determination of the acetate ion is important, especially in the areas of research involving kidney dialysis. However, known techniques are not subject to rapid and repeatable results with relatively inexpensive equipment.
A technique has been described for the determination of acetyl residues in protein as set forth in an article by Kuo et al entitled "A specific Micromethod for Determination of Acetyl Residues in Proteins", Analytical Biochemistry, 55, 1-8 (1973). This paper describes cleavage of the acetyl group from the protein under pressure in a wet chemical hydrolysis reaction. This acetate ion is then reacted with ATP in the presence of AK to form acetyl phosphate and ADP. The ADP is reacted with phosphoenolpyruvate (PEP) in the presence of pyruvate kinase (PK) to form ATP + pyruvate. The latter compound is then reacted with reduced nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase (LDH) enzyme. The amount of reduced NADH (colored) converted to oxidized nicotinamide adenine dinucleotide (NAD.sup.+) (uncolored) is detected optically to determine the quantity of acetate ion present in the product of hydrolysis. There is no disclosure that the above procedure could be employed for the detection of acetate ion present as such in a biological fluid such as serum or in a fluid such as wine or other fruit juice based products.
The technique disclosed in the Kuo et al. paper is subject to a number of significant disadvantages. One is that the reaction to form acetyl phosphate and ADP is relatively slow resulting in a total time on the order of 60 to 90 minutes. This is an obvious economic disadvantage when a large number of samples are to be analyzed by a technician. Furthermore, since the enzymic reactants are not totally stable, relatively long reaction times can be accompanied by deterioration to cause a lack of repeatability of the analytical results.